Figure 7. Clonal analyses of ILC1-like cells reveal high frequency of NKPs developing into cytotoxic KIR⁺NKG2A^- NK cells.

Single cells from the four ILC1-like subsets CD5⁺, CD161⁺, and CD161⁺CD5⁺ were flow cytometrically deposited in 96 well plates for clonal differentiation cultures on OP9-DL1 stroma cells. (a) Efficiency of clone growth at day 14. (b) Exemplary gating strategy for in vitro differentiated clones at day 28: CD94⁺NKG2A⁺ cells as well as CD94^-NKG2A^- cells were further divided on the basis of their respective NKG2A and KIR expression (upper panel). Pie charts and corresponding frequency of clones for the four different subsets (bottom panel): NKG2A⁺KIR^- (grey), NKG2A⁺KIR⁺ (black), NKG2A^-KIR^- (white), and NKG2A^-KIR⁺(blue) (n = 20 for all ILC1-like subsets, n = 4 for CD56^bright NK cells). (c) Quantification of CD107a cytotoxic mobilization assay with K562 cells in an effector/target ratio of 1:1 from single cell cultures (n = 3). (d) Representative microscopic picture from single cell culture exhibiting erasure of feeder cells by developing NK cells in the central region of the well. The heights of the bars represent the mean ± SEM. Levels of significance were calculated with a One-Way ANOVA with a multiple correction post-test (Kruskal-Wallis test). Data were generated from 288 CD5⁺ and CD161⁺ cells each, 177 CD5⁺CD161⁺ cells, and 12 CD56^bright NK cells sorted from a single donor.